Abstract: Acute Myelogenous Leukemia (AML) is a bone marrow-derived malignancy and remains a clinical challenge, with long-term survival of approximately 50%. Improvements in AML therapy have predominantly come through better supportive care and the improvements in allogeneic bone marrow transplantation, rather than improved chemotherapy approaches. This plateauing in AML survival points to a need for better, targeted therapies. Part of the difficulty with developing novel treatments to AML is that it is a heterogeneous disease, with well over 250 different mutated genes, although any one patient has on average 13 somatic mutations. Thus, developing a ?unifying genetic signature? that drives AML remains elusive. To understand how mutations found in AML contribute to leukemia development, we have chosen to focus on mutations in genes encoding the cohesin complex. Cohesin mutations occur in approximately 10-20% of patients, but its role in leukemogenesis is unknown. Our preliminary data demonstrate that cohesin loss causes increased self- renewal in wild-type murine bone marrow, a hallmark of leukemia development. Interestingly, this increased self-renewal is augmented in Npm1+/- after cohesin loss. While Npm1 mutations are the most common single gene mutation in AML, Npm1-mutated mice rarely develop AML, implying additional mutations are required. In both wild-type (WT) and Npm1-mutated bone marrow, cohesin loss causes epigenetic derepression of the self- renewal associated transcription factor HoxA9. Given that the Polycomb Repressive Complex 2 (PRC2), which mediates trimethylation of Histone 3 on Lysine 27 (H3K27me3), normally silences HoxA9 epigenetically we hypothesize that cohesin haploinsufficiency promotes AML by derepressing PRC2 target genes. The goal of this proposal is to use a combination of genome-wide approaches and genomic editing to determine the mechanism by which cohesin mutations induce abnormal self-renewal and promote leukemia development. In Aim 1 we will use chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) to identify changes in PRC2 genomic localization after cohesin loss. In Aim 2, we will use circular chromosomal conformational capture (4C) to identify which distal cis-regulatory elements (CREs) are normally recruited to regulate HoxA9 expression, and the changes in distal CREs after cohesin loss. In Aim 3 we will further characterize both in vitro and in vivo the effects of cohesin depletion on WT and Npm1cA/+ bone marrow. Also in Aim 3, we will test the role of the histone methyltransferase Dot1l loss/inhibition in preventing HoxA9 expression and cohesin-depletion associated self-renewal. Collectively, the long-term goal of our studies is to develop a novel mouse model of AML based upon loss of cohesin in Npm1cA AML, a combination of mutations that represents a sizeable fraction of patients with AML. In addition, we will use this model to address mechanistic question regarding how altered targeting of epigenetic complexes can cause disordered gene expression and leukemogenesis.